G protein-mediated signaling system is the most universal mechanism of cell communication and is involved in almost all cellular processes. RGS (regulator of G protein signaling) proteins play diverse and crucial functions to regulate G protein mediated signaling pathways. Galphaq-mediated activation of PLCbeta (phospholipase C-beta) regulates critical processes in various tissues, including cardiovascular system or nervous system. However, the molecular mechanism of its signal transduction and its regulation by RGS proteins have been poorly understood, mainly due to the lack of method to prepare enough amount of recombinant Galphaq for biochemical studies. Recently we developed an improved purification method to prepare functional recombinant Galphaq in large scale. Using this method, we have successfully isolated the complex of Galphaq-GRK2-Gbetagamma and determined its crystal structure. This structure revealed that RGS domain of GRK2 interacts with Galphaq like an effector. It also provided the first view of how heterotrimeric G protein moves and forms a signaling complex after receptor activation at the membrane. In addition, we recently identified p63RhoGEF as an effector for Galphaq. In this proposal, based on these results, we plan to further characterize the molecular mechanism of the interactions of Galphaq with RGS proteins, GRK2, p63RhoGEF, or PLCbeta. In Aim 1, we will isolate various Galphaq mutants that have specific defect to interact with RGS protein with GAP activity, GRK2, PLCbeta or p63RhoGEF. In Aim 2, using these mutants, we will characterize the physiological function of Galphaq-RGS/GRK2 interaction in receptor-mediated responses in cell. We also plan to apply SPR (surface plasmon resonance) method to analyze the kinetics of Gaphaq-RGS/GRK2 interaction with Galphaq-immobilized biosensor chip. We will also elucidate the molecular mechanism of Galphaq-RGS interaction by X-ray crystallography. In Aim 3, we will attempt to further improve PLCbeta stimulating activity of our recombinant Galphaq. We will apply SPR method to further understand the kinetics and thermodynamics of Galphaq-PLCbeta interaction. We also aim to determine the structure of Galphaq-PLCbeta3 complex by X-ray crystallography. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This proposal aims to understand the molecular mechanism of cells to respond to extracellular signals. The study will help to advance our understanding of various physiological functions. It will also contribute to develop novel drugs in future. [unreadable] [unreadable] [unreadable]